Abstract

Research on renewable biofuels produced by microorganisms has enjoyed considerable advances in academic and industrial settings. As the renewable ethanol market approaches maturity, the demand is rising for the commercialization of more energy-dense fuel targets. Many strategies implemented in recent years have considerably increased the diversity and number of fuel targets that can be produced by microorganisms. Moreover, strain optimization for some of these fuel targets has ultimately led to their production at industrial scale. In this review, we discuss recent metabolic engineering approaches for augmenting biofuel production derived from alcohols, isoprenoids, and fatty acids in several microorganisms. In addition, we discuss successful commercialization ventures for each class of biofuel targets.

@article{osti_1437002,
title = {Metabolic Engineering for Advanced Biofuels Production and Recent Advances Toward Commercialization},
author = {Meadows, Corey W. and Kang, Aram and Lee, Taek S.},
abstractNote = {Research on renewable biofuels produced by microorganisms has enjoyed considerable advances in academic and industrial settings. As the renewable ethanol market approaches maturity, the demand is rising for the commercialization of more energy-dense fuel targets. Many strategies implemented in recent years have considerably increased the diversity and number of fuel targets that can be produced by microorganisms. Moreover, strain optimization for some of these fuel targets has ultimately led to their production at industrial scale. In this review, we discuss recent metabolic engineering approaches for augmenting biofuel production derived from alcohols, isoprenoids, and fatty acids in several microorganisms. In addition, we discuss successful commercialization ventures for each class of biofuel targets.},
doi = {10.1002/biot.201600433},
journal = {Biotechnology Journal},
number = 1,
volume = 13,
place = {United States},
year = {2017},
month = {7}
}

For US transportation fuel independence or reduced dependence on foreign oil, the Federal Government has mandated that the country produce 36 billion gallons (bg) of renewable transportation fuel per year for its transportation fuel supply by 2022. This can be achieved only if development of efficient technology for second generation biofuel from ligno-cellulosic sources is feasible. To be successful in this area, development of a widely available, renewable, cost-effective ligno-cellulosic biomass feedstock that can be easily and efficiently converted biochemically by bacteria or other fast-growing organisms is required. Moreover, if the biofuel type is butanol, then the existing infrastructure tomore » deliver fuel to the customer can be used without additional costs and retrofits. The Claflin Biofuel Initiative project is focused on helping the US meet the above-mentioned targets. With support from this grant, Claflin University (CU) scientists have created over 50 new strains of microorganisms that are producing butanol from complex carbohydrates and cellulosic compounds. Laboratory analysis shows that a number of these strains are producing higher percentages of butanol than other methods currently in use. All of these recombinant bacterial strains are producing relatively high concentrations of acetone and numerous other byproducts as well. Therefore, we are carrying out intense mutations in the selected strains to reduce undesirable byproducts and increase the desired butanol production to further maximize the yield of butanol. We are testing the proof of concept of producing pre-industrial large scale biobutanol production by utilizing modifications of currently commercially available fermentation technology and instrumentation. We have already developed an initial process flow diagram (PFD) and selected a site for a biobutanol pilot scale facility in Orangeburg, SC. With the recent success in engineering new strains of various biofuel producing bacteria at CU, it will soon be possible to provide other technical information for the development of process flow diagrams (PFD’s) and piping and instrumentation diagrams (P&ID’s). This information can be used for the equipment layout and general arrangement drawings for the proposed process and eventual plant. An efficient bio-butanol pilot plant to convert ligno-cellulosic biomass feedstock from bagasse and wood chips will create significant number of green jobs for the Orangeburg, SC community that will be environmentally-friendly and generate much-needed income for farmers in the area.« less

Drop-in biofuels that are 'functionally identical to petroleum fuels and fully compatible with existing infrastructure' are needed for sectors such as aviation where biofuels such as bioethanol/biodiesel cannot be used. The technologies used to produce drop-in biofuels can be grouped into the four categories: oleochemical, thermochemical, biochemical, and hybrid technologies. Commercial volumes of conventional drop-in biofuels are currently produced through the oleochemical pathway, to make products such as renewable diesel and biojet fuel. However, the cost, sustainability, and availability of the lipid/fatty acid feedstocks are significant challenges that need to be addressed. In the longer-term, it is likely that commercialmore » growth in drop-in biofuels will be based on lignocellulosic feedstocks. However, these technologies have been slow to develop and have been hampered by several technoeconomic challenges. For example, the gasification/Fischer-Tropsch (FT) synthesis route suffers from high capital costs and economies of scale difficulties, while the economical production of high quality syngas remains a significant challenge. Although pyrolysis/hydrothermal liquefaction (HTL) based technologies are promising, the upgrading of pyrolysis oils to higher specification fuels has encountered several technical challenges, such as high catalyst cost and short catalyst lifespan. Biochemical routes to drop-in fuels have the advantage of producing single molecules with simple chemistry. Moreover, the high value of these molecules in other markets such as renewable chemical precursors and fragrances will limit their use for fuel. In the near-term, (1-5 years) it is likely that, 'conventional' drop-in biofuels will be produced predominantly via the oleochemical route, due to the relative simplicity and maturity of this pathway.« less

There is enormous interest in developing renewable sources of liquid fuels because of depletion of fossil fuel reserves, dependence on foreign sources, and increasing atmospheric CO 2 levels. Algae produce neutral lipids that are readily converted into liquid fuels such as biodiesel or JP-8 equivalent, and are attractive sources because they are far more productive than plants (yielding 10 -100’s of time more lipid per land area), and can be grown on non-cultivatable land with non-potable (brackish or salt) water sources. Unicellular algae known as diatoms were the most thoroughly characterized species in the National Renewable Energy Laboratory’s Aquatic Speciesmore » Program, whose goal was to develop microalgae as renewable fuel sources. Lipid accumulation in microalgae is generally induced by nutrient limitation, which involves a change in environmental conditions. Intrinsic variability in cellular response to environmental changes prevents a high degree of control over the process. Nutrient limitation also inhibits biomass accumulation; therefore a tradeoff between high biomass and lipid production occurs. The goal of this project was to develop metabolic engineering approaches for diatoms to enable induction of lipid accumulation by controllable manipulation of intracellular processes rather than from external environmental conditions, and to manipulate carbon partitioning within the cell between lipid and carbohydrate synthesis to enable both abundant biomass and lipid accumulation. There were two specific objectives for this project; Objective 1:To perform comparative transcriptomic analysis in T. pseudonana and C. cryptica of lipid accumulation resulting from silicon and nitrogen limitation, to identify common and key regulatory steps involved in controlling lipid accumulation and carbon partitioning; and Objective 2: To metabolically engineer the cell to alter carbon partitioning to either trigger lipid induction without the need for nutrient limitation, or to enable lipid accumulation along with high biomass accumulation.The significance of this project is that it will enable greater control over lipid production in diatoms by manipulable intracellular processes rather than from variable environmental conditions, and it will possibly enable lipid accumulation under normal growth conditions. Current economics dictate the use of open outdoor raceway pond systems for commercial-scale microalgal growth for biofuels production (although advanced design enclosed bioreactors are under consideration, they are currently not cost effective). Outdoor systems are subject to large variability in environmental conditions. In microalgae, lipid accumulation generally occurs under nutrient limiting conditions, which prevents high biomass accumulation. Potentially, one could carefully adjust the level of a particular nutrient so that it would become limiting after sufficient biomass accumulated; however, given the variability inherent in microalgal cellular metabolism under different light, temperature, and nutrient regimes, this will be a relatively uncontrolled and poorly reproducible approach. A better strategy would be to provide ample nutrients, but trigger lipid accumulation “artificially” by manipulating intracellular processes through metabolic engineering. In addition, identifying the key regulatory steps involved in controlling carbon partitioning in the cell coupled with metabolic engineering should enable greater partitioning of carbon into lipids during non-limiting nutrient growth conditions. The approaches outlined in this proposal are aimed at achieving these goals, and are expected to have a substantial impact on the development of renewable biofuels technology. Development of the approaches described in this proposal will provide a rich interdisciplinary educational experience for high school and undergraduate students to foster their development in a scientific career.« less

During the past ten years, there has been significant interest and investment in the study of catalytic conversion of biomass-derived feedstocks into renewable fuels and chemicals. In the United States, an estimated $25 billion has been spent by venture capitalists, industry, and government agencies during this period of time to commercialize “renewable technologies” including solar energy, wind power, batteries, and biofuels (e.g., cellulosic ethanol). Four societal factors are driving these investments including: (1) the increased price of crude oil; (2) concerns about global warming; (3) the desire to improve rural economies where biomass is produced, and; (4) national goals tomore » become energy self-sufficient. Furthermore, underlying these efforts is the realization that lignocellulosic biomass is the only realistic, near-term and non-food-competitive source of renewable organic carbon. To this end, legislative efforts, such as the US Renewable Fuel Standards, have been implemented to create subsidies, tax credits, mandates, and loan guarantees to help bring renewable fuel technologies to market. However, the representative body of industrial efforts to this end has faced significant challenges, with several startup companies having commercialized biomass conversion technologies but having struggled to reach commercial scale; in fact, many of these companies have filed for bankruptcy. This situation is likely due to the challenges associated with scaling up unproven pioneer processes, as well as neophyte investors not understanding the decade-long time frames and the sheer amount of funding that is often required to bring chemical process technologies to market. Nevertheless, several emerging catalytic technologies have either entered the market place, or are currently demonstrating their technologies in fully integrated pilot plants. Commercial and near commercial technologies for second generation biomass conversion technologies to-date include, among others: biomass-derived jet and diesel fuel from both waste vegetable oils and ethanol; small scale production of renewable jet and diesel from landfill gases via Fischer-Tropsch synthesis; catalytic conversion of carbohydrates into gasoline and aromatics; hydropyrolysis of biomass into gasoline and diesel, and; catalytic conversion of wood into aromatics. To be successful, biomass conversion technologies must ultimately be able to compete economically with petroleum technologies, which are already operating at large commercial scales and have been practiced for decades. To this end, various factors must be considered when evaluating the potential for new processes to compete with incumbent technologies; factors such as regional variations in feedstock quality and availability, government policy, subsidies and tax rates, and the proprietary positions of the ancillary technologies that might support the process. By any measure, however, a critical metric of the economic potential of a process is its efficiency with respect to the yield of products from raw materials, and this metric of performance is directly related to atom efficiency of the underlying chemical reactions. Therefore, while promising biomass conversion technologies continue to demonstrate progress towards commercialization, there remains an important need for the catalysis community to aid in this effort by: (1) designing more active, selective and stable catalysts, and (2) elucidating a more detailed understanding of the catalytic chemistries underlying these processes. Indeed, the study of catalytic biomass conversion has grown tremendously during the past decade; and new or emerging technologies that are in the laboratory stage have allowed for biomass to be converted into a wider variety of commodity chemicals and the full range of liquid fuels that are produced from petroleum. The objective of this perspective is to highlight some of the ongoing, fundamental challenges with respect to the catalytic conversion of biomass into renewable products, and to provide insight as to how the catalysis community can overcome several of these challenges. It is our belief that, as an international academic community of catalysis researchers, we can (and must) work together to address these challenges, and to drive progress toward the production of next-generation renewable chemicals and fuels from biomass.« less

Microalgae represent an exceptionally diverse but highly specialized group of micro-organisms adapted to various ecological habitats. Many microalgae have the ability to produce substantial amounts (e.g. 20-50% dry cell weight) of triacylglycerols (TAG) as a storage lipid under photo-oxidative stress or other adverse environmental conditions. Fatty acids, the building blocks for TAGs and all other cellular lipids, are synthesized in the chloroplast using a single set of enzymes, of which acetyl CoA carboxylase (ACCase) is key in regulating fatty acid synthesis rates. However, the expression of genes involved in fatty acid synthesis is poorly understood in microalgae. Synthesis and sequestrationmore » of TAG into cytosolic lipid bodies appear to be a protective mechanism by which algal cells cope with stress conditions, but little is known about regulation of TAG formation at the molecular and cellular level. While the concept of using microalgae as an alternative and renewable source of lipid-rich biomass feedstock for biofuels has been explored over the past few decades, a scalable, commercially viable system has yet to emerge. Today, the production of algal oil is primarily confined to high-value specialty oils with nutritional value, rather than commodity oils for biofuel. This review provides a brief summary of the current knowledge on oleaginous algae and their fatty acid and TAG biosynthesis, algal model systems and genomic approaches to a better understanding of TAG production, and a historical perspective and path forward for microalgae-based biofuel research and commercialization.« less